When blood escapes from the vasculature, an intricate cascade of enzymatic reactions converts fibrinogen to fibrin, the structural protein in clotted blood. Blood clots, also called thrombi, may also be inappropriately formed within blood vessels in certain pathological conditions. Fibrinogen itself is the least soluble of the plasma proteins. With a 340,000 kDa MW, it possesses a two-fold symmetry arising from three pairs of non-identical polypeptide chains called A-alpha, B-beta and gamma. At the site of thrombosis, the coagulation cascade is activated to generate thrombin, which enzymatically cleaves polar peptides (Fibrinopeptide A from A-alpha and Fibrinopeptide B from B-beta), and results in fibrin monomer formation. Fibrin monomers, being much less soluble, spontaneously polymerize into a gel network. After polymerization, the fibrin clot is stabilized by Factor XIIIa, which introduces covalent interchain .epsilon.-(.gamma.-glutamyl)lysine bonds. Fibrinogen and fibrin are identical in greater than 98% of their structure and differ only in two newly exposed amino termini, those of the fibrin alpha and beta chains. The amino acid sequence of these fibrin amino termini is known (Doolittle, R. F., "Fibrinogen and Fibrin," in Putnam, F. W., ed., The Plasma Proteins: Structure, Function, and Genetic Control, 3d ed., Vol. 2, New York: Academic Press, 1975, pp. 109-156).
Most myocardial infarctions are caused by a coronary thrombosis (DeWood et al., N. Eng. J. Med. 303:897 (1983)). The coronary thrombus can be lysed by thrombolytic agents thus restoring blood flow to the affected portion of the heart. These thrombolytic agents may be thrombolytic enzymes such as a plasminogen activator (PA). The PAs activate the conversion of plasminogen to the fibrinolytic enzyme plasmin. Plasmin has an affinity for fibrin and will lyse the fibrin present in the thrombus. This treatment with PAs is not without side effects. Plasmin acts non-selectively and therefore not only lyses the fibrin in the thrombus, but also attacks fibrinogen and clotting factors often resulting in severe bleeding diathesis.
Streptokinase, urokinase, prourokinase, and tissue-type PA (tPA) are PAs used for lysing thrombi. These PAs are indicated for the treatment for acute cardiovascular disease such as infarct, stroke, pulmonary embolism, deep vein thrombi, peripheral arterial occlusion, and other venous thrombi.
Streptokinase and urokinase constitute the first generation of PAs. Both streptokinase and urokinase, however, have severe limitations. Due to a low affinity for fibrin, both PAs will activate circulating and fibrin-bound plasminogen indiscriminately. The plasmin formed in circulating blood is neutralized before it can be used in thrombolysis. Residual plasmin will degrade several clotting factor proteins, for example, fibrinogen, factor V, and factor VIII, causing hemorrhagic potential.
Streptokinase, a bacterial protein, was the first identified PA. It forms a 1:1 stoichiometric complex with plasminogen and thereby converts it to its active form, plasmin. When administered within 4 hours of coronary occlusion, streptokinase has been shown to reduce mortality after myocardial infarction in a number of randomized trials (Simoons et al., J. Am. Coll. Cardiol. 7:717 (1986); and Hartman et al., Am. Heart J. 111:1030 (1986)). However, the use of this agent is invariably accompanied by a marked depletion of fibrinogen caused by the generation of excess plasmin. Further, streptokinase is strongly antigenic and patients with high antibody titers against it respond inefficiently to treatment and cannot remain on continuous treatment.
Urokinase is a two-chain, trypsin-like serine protease that activates. plasminogen by limited proteolysis of the single, specific Arg-560-Val peptide bond (Violand et al., J. Biol. Chem. 251:3906-3912 (1976)). Results obtained with urokinase have been similar to those obtained for streptokinase in smaller-scale clinical trials (Mathey et al., Am. J. Cardiol. 55:878 (1985)).
Second generation PAs include tPA and single chain urokinase-like PA (scuPA). Unlike streptokinase and urokinase, tPA and scuPA exhibit fibrin-selective plasminogen activation. The selectivity of tPA derives from the presence of a fibrin binding site on the molecule. tPA binds fibrin with a kDa of 0.16 .mu.M; when bound, its K.sub.m for plasminogen activation decreases from 83 .mu.M to 0.18 .mu.M and its k.sub.cat increases from 0.07 to 0.28 sec.sup.1, resulting in an increase in catalytic efficiency of approximately 1000 fold. Although scuPA probably does not bind directly to fibrin, it activates fibrin-bound plasminogen much more readily than plasma plasminogen. Its fibrin selectivity is comparable to that of tPA (Collen et al., Thromb. Haemost. 52:27 (1984)).
tPA and scuPA are also considered native PAs because endothelial and other cells secrete them into the circulation. Initial studies of tPA and scuPA were conducted on proteins purified from cultured cell lines including the Bowes melanoma cell line for tPA, and transformed human kidney cells for scuPA. Both agents have subsequently been produced by recombinant DNA methods (Pennica et al., Nature 301:214 (1983); Holmes et al., Biotechnology 3:923 (1985)).
scuPA is cleaved by plasmin between amino acids Lys 158 and Ile 159. The resulting high molecular weight (HMW) two-chain urokinase has the catalytic activity of scuPA but does not have the fibrin selectivity and resistance to plasminogen activator-inhibitor I of its single-chain precursor. Low molecular weight (LMW) two-chain urokinase is the first generation form. The full length, HMW form of scuPA is the native PA and is the form that has been studied clinically as a second generation PA.
The light chain (amino terminal) of scuPA contains, in addition to an epidermal growth factor-like domain, a single kringle region that shows considerable homology with the kringles of tPA, despite the fact that scuPA does not appear to bind fibrin. Another property that differentiates scuPA from tPA is scuPA's resistance to irreversible inhibition by plasminogen activator-inhibitor I, as well as to other plasminogen activator-inhibitors. For this reason, unlike tPA, scuPA is stable in human plasma for extended periods. For example, plasminogen activator-inhibitor I binds reversibly to scuPA: when scuPA forms a ternary complex with fibrin and plasminogen, plasminogen activator-inhibitor I is displaced. It is not until after plasmin cleaves scuPA between residues Lys 158 and Ile 159 to form HMW two-chain urokinase that the catalytic site becomes susceptible to irreversible inhibition. LMW two-chain urokinase derives from subsequent cleavage of the Lys 136-Lys 137 peptide bond and is readily inhibited by plasminogen activator-inhibitor I.
Stump et al. (J. Biol. Chem. 261:17120 (1986)) have described a shortened form of scuPA that results from proteolytic cleavage during purification between residues Glu 143 and Leu 144. scuPA is probably not present in this form in vivo. LMW scuPA, now expressed by recombinant DNA methods (Nelles et al., J. Biol. Chem. 262:10855 (1987)) does not contain the amino terminal kringle.
Although it is only 14 amino acids longer than LMW two-chain urokinase, LMW scuPA manifests fibrin selectivity identical to that of native, HMW scuPA, clearly excluding the kringle from a role in fibrin selectivity. LMW (32-kDa) scuPA also retains another important property of native (54-kDa) scuPA--its resistance to plasminogen activator-inhibitor I.
Human tPA binds to fibrin and therefore favors the activation of plasminogen in close proximity to the thrombus thus potentially sparing fibrinogen elsewhere in the circulation. However, at the doses required for prompt lysis of coronary thrombi, the use of tPA can also result in hemorrhage.
It is now established that early therapy with PAs reduces mortality in patients with acute myocardial infarction (Aims Trial Study Group, Lancet I:545-549 (1988); Check, W. A., Clin. Pharm. 10:486-7 (1991); GISSI, Lancet I:397 (1986); GISSI-2, Lancet 336:65-71 (1990); ISIS-3, Lancet 339:753-770 (1992); and Simoons et al., Lancet II:578-582 (1985)). However, this treatment is complicated by: a) failure to achieve reperfusion in 15-20% of patients (TIMI Study Group, New Engl. J. Med. 312:932-936 (1985); Topol et al., Ann. Intern. Med. 103:837-843 (1985); Topol et al., Circulation 77:1100-1107 (1988)); b) abnormal bleeding, particularly hemorrhagic stroke in .about.0.5 to 1% of the patients, that requires blood transfusions in .about.10% of the patients (ISIS Steering Committee, Lancet 1987-I:502 (1987); Holvoet et al., J. Biol. Chem. 266:19717-19724 (1991)); and c) rethrombosis after cessation of therapy in 5-15% of patients (Topol et al., J. Am. Coll. Cardiol. 9:1205-1213 (1987); Topol et al., N. Engl. J. Med. 317:581-588 (1987); Chesebro et al., N. Engl. J. Med. 319:1544-1545 (1988); Braunwald et al., J. Clin. Invest. 76:1713-1719 (1985)). To reduce these complications, PAs have been developed that exhibit increased specificity for thrombus, altered clearance properties, or reduced inactivation by plasma inhibitors (Jackson et al., Circulation 82:930-40 (1990); Runge et al., Proc. Natl. Acad. Sci. USA 88:10337-10341 (1991); Collen et al., Circulation 82:1744-1753 (1990); Lijnen et al., J. Biol. Chem. 263:5594-5598 (1988); Browne et al., J. Biol. Chem. 263:1599-1602 (1988); Madison et al., Nature 339:721-724 (1989); Nelles et al., J. Biol. Chem. 262:5682-5689 (1987)). Initial studies in animal models demonstrated modest improvements in thrombolytic efficacy for most of these "third generation" molecules. In addition, PA therapy has been combined with therapeutic agents that inhibit platelet function or reduce thrombin activity (Heras et al., Circulation 79:657-665 (1989); Eidt et al., J. Clin. Invest. 84:18-27 (1989); Jang et al., Circulation (Suppl) 80:II:217 (1989)). While combined therapies augment thrombolysis and decrease rethrombosis, they also increase the risk of bleeding because they interrupt hemostatic function.
In order to increase the specificity of the thrombolytic agents to the thrombus, it has been shown that covalent linkage of urokinase to a fibrin-specific monoclonal antibody results in marked enhancement of fibrinolytic potency and specificity (Bode et al., Science 229:765-767 (1985)).
One function which is characteristic of every antibody molecule is specific binding to an antigenic determinant. Antibodies in vivo are bivalent and monospecific, containing two identical antigen binding sites. The specificity of the binding of an antigen by an antibody molecule is determined by the structure of the antibody's variable regions (F.sub.ab) of both heavy and light chains.
Antibodies are tetrameric immunoglobulins consisting of two identical light (L) chains and two identical heavy (H) chains. Each protein chain consists of two principle regions: the N-terminal variable (V) region and the C-terminal constant (C) region. The variable light (V.sub.L) and heavy (V.sub.H) chains form the variable region domain. The variable domain determines recognition and specificity to a particular antigen. The constant region domains of light (C.sub.L) and heavy (C.sub.H) chains mediate the effector function responsible for executing the immune response. The hinge region (J) of the antibody molecule connects the Fab fragment to the Fc fragment of the antibody.
Within the variable region, there may be hypervariable regions known as diversity domains (D). These diversity domains are related to exons observed in the genes encoding for the variable regions.
The variable domain of an antibody, a protein structural definition, consists of both V.sub.L and V.sub.H segments of the light and heavy chains. It contains 6 hypervariable regions, three in the light chain and three in the heavy chain. On a genetic level, three exons are responsible for specifying V.sub.H, including its framework and hypervariable regions; two exons specify V.sub.L. The first two hypervariable regions of both V.sub.L and V.sub.H are specified by the V gene exons of the light and heavy chains respectively. The third hypervariable region of the light chain is specified by two exons, V.sub.L and J.sub.L. The third hypervariable region of the heavy chain is specified by three exons V.sub.H, D, and J.sub.H.
Immunoglobulin gene expression occurs through the joining of the V gene to the C gene by somatic recombination in the B lymphocytes. These genes are joined to form the complete immunoglobulin. The rearranged, joined gene segments then encode the complete immunoglobulin or antigen binding domains of light and heavy variable chains.
There are five principal classes of heavy chains, characterized by chemical and isotypic properties. These heavy chain classes are referred to as mu, gamma, delta, alpha, and epsilon. There are five principal classes of immunoglobulins (antibodies) referred to as: IgG; IgM; IgD; IgA; and IgE. There are also two principal classes of light chains: kappa and lambda.
Antibodies with specificity to fibrin have been described in Hui et al. (Science 222:1129 (1983)). Other examples of antibodies with a specificity against fibrin have been described (Kudryk et al., Mol. Imm. 21:89 (1984); European Patent Application 146,050 to Callewaert, published Jun. 26, 1985, for "Site Selective Plasminogen Activator and Method of Making and Using Same"; and Australian Patent Application, AV-A-25387/84 to Bundesen et al. for "Monoclonal Antibodies with Specificity for Crosslinked Fibrin and Their Diagnostic Uses").
Antibodies having dual specificities have been prepared by subjecting antibodies of different specificities to a selective cleavage of the disulfide bridges that link the two heavy chains together. Antibody half-molecules are then reassociated under neutral pH to produce the hybrid antibodies having dual specificities (see, for example, Nisonhoff et al., Nature (London) 394:355 (1962); Brennan et al., Science 229:31 (1985); Liu et al., Proc. Natl. Acad. Sci. USA 82:8648 (1985); and commonly assigned formerly United States patent application, Ser. No. 851,554, filed Apr. 14, 1986, now U.S. Pat. No. 4,916,070).
Bispecific antibodies have also been produced from hybridomas. The preparation of bispecific monoclonal antibodies by fusion of antibody-producing hybridoma cells is described in Milstein et al., Nature (London) 305:537 (1983) and in PCT application WO83/103679.
Antibodies have also been cloned and produced by recombinant DNA techniques. Genes for heavy and light chains have been introduced into appropriate hosts and expressed, followed by reaggregation of these individual chains into functional antibody molecules (see, for example, Munro, Nature 312:597 (1984); Morrison, S. L. Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986)); Wood et al., Nature 314:446-449 (1985)). Light and heavy chain variable regions have been cloned and expressed in foreign hosts, and maintain their binding ability (Moore et al., European Patent Publication 0088994 (published Sep. 21, 1983)).
Chimeric or hybrid antibodies have also been prepared by recombinant DNA techniques. Oi and Morrison describe a strategy for producing chimeric antibodies (BioTechniques 4:214 (1986)). On pages 218-220 thereof a chimeric:human IgG anti-Leu3 antibody is described. The authors state that a chimeric mouse:human anti-dansyl antibody has been made. This article indicates, without specifically stating so, that the Leu3 binding specificity and the anti-dansyl binding specificity have been cloned together into a single immunoglobulin molecule.
Morrison states that variable light or variable heavy chain regions can be attached to a non-Ig sequences to create fusion proteins (Table 1, Science 229:1202 (1985)). This article states that the fusion proteins have three potential uses: (1) to attach antibody specificity to enzymes for use in assays; (2) to isolate non-Ig proteins by antigen columns; and (3) to specifically deliver toxic agents. There is no description in this reference of any specific chimeric immunoglobulin molecule.
Neuberger et al. describe a chimeric antibody whose heavy chain is a human constant region fused to a mouse variable region that is specific for the hapten, 4-hydroxy-3-nitrophenyl-acetyl (Nature 314:268 (1985)).
European Patent Application 120,694 describes the genetic engineering of the variable and constant regions of an immunoglobulin molecule that is expressed in E. coli host cells. The application states on page 10 that the immunoglobulin molecule may be synthesized by a host cell with another peptide moiety attached to one of the constant domains. This peptide moiety is either cytotoxic or enzymatic. It also states on page 10 that the immunoglobulin molecule may also comprise a therapeutic agent. The description in the application and in the examples describe the use of a lambda-like chain derived from a monoclonal antibody which binds to 4-hydroxy-3-nitropenylacetal (NP) haptens.
European Patent Application 125,023 relates to the use of recombinant. DNA techniques to produce immunoglobulin molecules that are chimetic or otherwise modified. One of the uses for these immunoglobulin molecules is their use in whole body diagnosis and treatment, wherein antibodies directed to specific target disease tissues are injected into a patient (pages 3-4). The presence of the disease can be determined by attaching a suitable label to the antibodies, or the diseased tissue can be attacked by carrying a suitable drug with antibodies. The application describes antibodies engineered to aid the specific delivery of an agent as "altered antibodies."
PCT application WO83/03971 relates to a hybrid protein that comprises antibody-enzymatically active toxins.
PCT application W083/01533 describes on page 5 chimetic antibodies with the variable region of an immunoglobulin molecule linked to a portion of a second protein which may comprise the active portion of an enzyme.
Boulianne et al. constructed an immunoglobulin gene in which the DNA segments that encode mouse variable regions specific for the hapten trinitrophenol (TNP) are joined to segments that encode human mu and kappa constant regions (Nature 312:643 (1984)). These chimeric genes were expressed as functional TNP-binding chimeric IgM.
Morrison et al. created a chimetic molecule utilizing the heavy chain variable region exons of an anti-phosphoryl choline myeloma protein gene, which were joined to the exons of either human kappa light chain gene (Proc. Natl. Acad. Sci. USA 81:6851 (1984)). The genes were transfected into mouse myeloma cell lines, generating transformed cells that produced chimeric mouse-human IgG with antigen binding function.
Sharon et al. fused a gene encoding a mouse heavy chain variable region specific for azophenylarsonate with the mouse kappa light chain constant region gene (Nature 309:604 (1984)). This construct resulted in a polypeptide chain that dimerized with the corresponding V.sub.L -Kappa polypeptide chain when introduced into the appropriate myeloma cell line. The V.sub.Hkappa V.sub.L C.sub.kappa molecule bound to the azophenylarsonate hapten.
Neuberger et al. joined the heavy chain variable region gene of a hapten-specific antibody to a gene specifying the synthesis of micrococcal nuclease, and obtained a hybrid molecule that had both antigen binding and enzymatic activity (Nature 312:604 (1984)).
Robbins and coworkers described covalently linked hybrid PAs that covalently linked the fibrin binding of the plasminogen A chain with the catalytic domain of urokinase (Biochemistry 25:3603-3611 (1986)). Stump et al. described a shortened form of scuPA which, like scuPA, was fibrin specific, although it apparently did not bind to fibrin directly (J. Biol. Chem. 26:17120-17126 (1986)). Attempts to further improve the fibrin specificity of this molecule by either site-directed mutagenesis to provide stability (Nelles et al., J. Biol. Chem. 262:10855-10862 (1987)), or by conferring direct fibrin affinity by creating a recombinant molecule combining the fibrin-binding A chain of tPA with the low molecular weight (LMW) form of scuPA (Nelles et al., J. Biol. Chem. 262:5682-5689 (1987) were disappointing.
It would be desirable to have a selective thrombolytic enzyme, such as a PA, that is characterized by high affinity and specificity for fibrin relative to fibrinogen, and that would effect activation of plasminogen only in the immediate environment of a fibrin-containing thrombus.